The LLC-PK, cell line, which was originally derived from pig kidney, is a widely used model system for studying the structure and physiological properties of renal transporters. During the past grant period, we have discovered that LLC-PKI cells express two distinct forms of the Na/H antiporter: NAH-1, found in rapidly growing cells and on the basolateral surface at confluence, and NAH-2, appearing on the apical surface at confluence. The two forms exhibit a 300-fold difference in sensitivity to ethylisopropylamiloride and are also affected independently by genetic mutations. During the next grant period, we will pursue several main goals. Starting with the NAH-1 gene, which we have already cloned and sequenced, we will attempt to isolate the gene for NAH-2 by low-stringency hybridization. Alternative strategies have been designed in the event that this straightforward approach is not successful. As soon as both genes are in hand, specific DNA probes and antibodies will be developed to track the synthesis and localization of NAH-1 and NAH-2 during differentiation, both in the normal LLC-PK,/Clone 4 cell line and in two existing mutants. Additional antiporter-deficient mutants will be isolated and characterized to obtain information about structure-function relationships. And finally, the cloned NAH-1 and NAH-2 genes will be expressed in antiporter-deficient cells and/or in yeast to confirm the physiological properties of the two antiporters and to pave the way for site-directed mutagenesis. Taken together, the results of this work should fumish useful new information about the structure, biogenesis, and transport mechanism of Na/H antiporters in the kidney.